biological hydrogen production using ......biological hydrogen production using organic waste and...
TRANSCRIPT
INTEGRATED MASTER IN ENVIRONMENTAL ENGINEERING 2012/2013
BIOLOGICAL HYDROGEN PRODUCTION USING ORGANIC WASTE
AND SPECIFIC BACTERIAL SPECIES
ANA RAQUEL RIBAS CORDEIRO
Dissertation submitted for the degree of
MASTER ON ENVIRONMENTAL ENGINEERING
President of the jury: Professor Doctor Cidália Maria de Sousa Botelho Assistant Professor at the Department of Chemical Engineering of the Faculty of
Engineering of the University of Porto (FEUP)
_______________________________________________________________
Supervisor at the hosting institution: Professor Raffaello Cossu Head of the Environmental Engineering Programme at the Department of Civil, Environmental and Architectural Engineering (ICEA) of the University of Padova
Co – Supervisor at the hosting institution: Doctor Luca Alibardi Research Fellow at the Department of Civil, Environmental and Architectural
Engineering (ICEA) of the University of Padova
October 2013
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
ACKNOWLEDGEMENTS
I take here the opportunity to thank Professor Cossu to have given me the opportunity to
perform my thesis abroad and to be part of a fantastic team work in the Laboratory of
Environmental and Sanitary Engineering at the Department of Civil, Environmental and
Architectural Engineering of the University of Padova.
I also would like to thank Alessandra Ruzza and Mubashir Saleem to have divided this
experiment with me, for the team work that we shared together and for helping me in
the moments that I most needed.
I would like to thank Annalisa Sandon, the technician of the laboratory, first of all
because she taught me how to perform the chemical analyses with a lot of patience and
also for her availablility for my questions and sample analysis.
My special thanks to Doctor Luca, to have strictly followed me throughout my work
and also to have taught me everything that was necessary for my research. His help was
undoubtedly essential for my success.
To Professor Cidália my gratitude for her willingness and help during ERASMUS
programme.
I would like to thank to all my friends and family for all the friendship and love, in
particular, a special thanks to my boyfriend for the unconditional support and love.
Finally a very special thanks to my mother, who is my pillar and motivation source for
all my life projects.
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
ABSTRACT
Hydrogen (H2) is a valuable gas required as feedstock for several industries that can also
be used as a clean energy source. Demand on hydrogen production has increased
considerably in recent years. Biological hydrogen production (BHP) appears to be very
promising as it is non-polluting process and hydrogen can be produced from water and
biodegradable wastes.
Dark fermentation is the biological process for hydrogen production showing the
highest potentials for sustainable hydrogen production, because of its high production
rates in the absence of a light source and thank to the possibility of using a variety of
different organic substrates. Furthermore, the efficiency of energy production can be
improved by screening microbial diversity and easily fermentable feed materials.
In this sense, the present study reports a research on biological hydrogen production
from a real mixed bacteria culture, represented by three specific types of granular
sludge, and from pure cultures constituted of five dark fermentative bacteria (Bacillus
licheniformis, Paenibacillus cookie, Bacillus sp., Paenibacillus sp. and Bacillus
farraginis). These hydrogen production tests were performed in batch reactors under
mesophilic conditions.
The highest hydrogen production was measured by the mixed bacteria culture,
represented by the sample “Sludge 2013”, with a total hydrogen production of 148.2
NmlH2/gVS. Among the five different fermentative bacteria, Bacillus farraginis showed
great performance with a total hydrogen production of 95.2 NmlH2 /gVS.
Furthermore, other results showed that the efficiency of the hydrogen production was
decreased by increasing glucose concentration. It was also proven that the most efficient
condition to obtain the higher hydrogen production is the addition of Nutrient Broth
(NB), in a first run, in order to provide the necessary nutrients for the bacteria, and
subsequently add glucose as carbon source.
Future researches may be interesting, in order to obtain an inoculum that is a consortium
of bacteria characterized by high potentials for hydrogen production for further scale-up
and industrial application.
Keywords: Hydrogen; Dark fermentation; Mixed cultures; Pure cultures; Bacillus.
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
INDEX
1. INTRODUCTION ................................................................................................................. 1
2. EXPERIMENTAL PROCEDURES ..................................................................................... 5
2.1 Research scheme ............................................................................................................. 5
2.2 Inoculum conditioning and characterization ................................................................... 5
2.2.1 Real mixed bacteria culture ................................................................................... 5
2.2.2 Batch mixed reactor .............................................................................................. 6
2.2.3 Pure bacteria culture .............................................................................................. 7
2.3 Batch test for hydrogen production ................................................................................. 9
2.3.1 Real mixed bacteria culture ................................................................................... 9
2.3.2 Batch mixed reactor ............................................................................................ 10
2.3.3 Pure bacteria culture ............................................................................................ 11
2.3.3.1 Experiment nº 1 ........................................................................................ 11
2.3.3.2 Experiment nº 2 ........................................................................................ 13
2.4 Methods ......................................................................................................................... 14
2.4.1 Analytical methods .............................................................................................. 14
2.4.2 Experimental data results .................................................................................... 14
2.4.3 Mathematical models of hydrogen production .................................................... 15
3. RESULTS AND DISCUSSION .......................................................................................... 17
3.1 Biological hydrogen potential production ..................................................................... 17
3.1.1 Real mixed bacteria culture ................................................................................. 17
3.1.2 Batch mixed reactor ............................................................................................ 24
3.1.3 Pure bacteria culture ............................................................................................ 25
3.1.3.1 Experiment nº 1: “Run 1” ......................................................................... 26
3.1.3.2 Experiment nº 1: “Run 2” ......................................................................... 29
3.1.3.3 Experiment nº 2: “Run 1” ......................................................................... 33
3.1.3.4 Experiment nº 2: “Run 2” ......................................................................... 35
3.2 Process performance ...................................................................................................... 37
3.2.1 Overall evaluation of the results obtained by the pure cultures .......................... 37
4.2.2 Evaluation of the potential hydrogen productions from the mixed and the pure
bacteria culture ............................................................................................................... 43
4. CONCLUSIONS ................................................................................................................. 45
REFERENCES ............................................................................................................................ 47
ANNEX 1 – Results of the BMP tests ........................................................................................ 51
i
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
LIST OF FIGURES
Figure 1 – Cumulative hydrogen productions from average experimental data and from the
mathematical model by the samples: (I) “Sludge 2011”; (II) “Sludge 2012”; (III) “Sludge
2013”. The vertical bars over the experimental data represent the standard deviations of the
triplicate....................................................................................................................................... 18
Figure 2 – Comparison between the cumulative hydrogen productions from the mathematical
model, by the samples “Sludge 2011”, “Sludge 2012” and “Sludge 2013” ................................ 19
Figure 3 – Volatile fatty acids composition of dark fermentation the samples “Sludge 2011”,
“Sludge 2012” and “Sludge 2013” .............................................................................................. 21
Figure 4 – Cumulative hydrogen consumption from average experimental data by the samples:
(I) “Sludge 2011”; (II) “Sludge 2012”; (III) “Sludge 2013”. The vertical bars over the
experimental data represent the standard deviations of the triplicate .......................................... 22
Figure 5 – Comparison between the cumulative hydrogen consumptions, by the samples
“Sludge 2011”, “Sludge 2012 and “Sludge 2013”, and their trend lines .................................... 22
Figure 6 – Comparison between the hydrogen production rates, by the samples “S_40”, “S_20”
and “S_10” .................................................................................................................................. 24
Figure 7 – Optical Density and pH variations, over time, and cumulative hydrogen productions
from average experimental data and from the mathematical model by the samples: (A) B.
licheniformis; (B) P. cookie; (C) Bacillus sp. The vertical bars over the experimental data
represent the standard deviations of the triplicate ....................................................................... 27
Figure 8 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A2 and A3 ................................................................................ 30
Figure 9 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples B2 and B3 ................................................................................ 31
Figure 10 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples C2 and C3 ................................................................................ 31
Figure 11 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A2, B2 and C2 and by the samples A3, B3 and C3, with: (I) 5
g/l of glucose; (II) 10 g/l of glucose ............................................................................................ 32
Figure 12 – Optical Density and pH variations, over time, and cumulative hydrogen
productions, in experiment nº2 “Run 1”, from average experimental data and from the
mathematical model by the samples: (A) B. licheniformis; (B) P. cookie; (C) Bacillus sp; (D)
Paenibacillus sp.; (E) Bacillus farraginis. The vertical bars over the experimental data represent
the standard deviations of the triplicate ....................................................................................... 34
Figure 13 – Optical Density and pH variations, over time, and cumulative hydrogen
productions, in experiment nº2 “Run 2”, from average experimental data and from the
mathematical model by the samples: (A) B. licheniformis; (B) P. cookie; (C) Bacillus sp; (D)
Paenibacillus sp.; (E) Bacillus farraginis. The vertical bars over the experimental data represent
the standard deviations of the triplicate ....................................................................................... 36
ii
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Figure 14 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A, B and C in the experiment nº 1 – “Run 1” .......................... 37
Figure 15 – Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A, B, and C in the experiment nº 2 – “Run 1” ......................... 38
Figure 16 – Hydrogen yields, from “Runs 1”, obtained by the experiment nº 1 and by the
experiment nº 2 ............................................................................................................................ 39
Figure 17 – Hydrogen yields, from “Runs 2”, obtained by the experiment nº 1 and by the
experiment nº 2 ............................................................................................................................ 40
Figure 18 – Cumulative hydrogen productions, from the mathematical model, obtained by the
mixed and pure bacteria cultures, which are “Sludge 2013” and Bacillus sp, respectively ........ 43
iii
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
LIST OF TABLES
Table 1 – Physical and chemical characterization of the granular sludge samples ...................... 6
Table 2 – Identification of the microbial strains used and their hydrolytic abilities .................... 7
Table 3 – Delineation of the experimental activities performed in batch test .............................. 8
Table 4 – Results from hydrogen production batch tests, obtained by each real mixed bacteria
culture sample ............................................................................................................................. 17
Table 5 – Mathematical model parameters, obtained by each real mixed bacteria culture sample
..................................................................................................................................................... 17
Table 6 – Results from hydrogen consumption batch tests, obtained by each real mixed bacteria
culture sample ............................................................................................................................. 23
Table 7 – Results from hydrogen yields and rates at different F/M ratios, from the mathematical
model, by each sample using the batch mixed reactor ................................................................ 24
Table 8 – Results from analytical analysis, by each sample using the batch mixed reactor ...... 25
Table 9 – Results from hydrogen production batch tests, for the experiment nº1–“Run 1” ....... 26
Table 10 – Mathematical model parameters, obtained by the sampled from the experiment nº1 –
“Run 1”........................................................................................................................................ 26
Table 11 – Results from hydrogen production batch tests, for the samples A2, B2 and C2 and
for the samples A3, B3 and C3, in the experiment nº 1 – “Run 2” ............................................. 29
Table 12 – Mathematical model parameters, for the sample A2, B2 and C2 and for the samples
A3, B3 and C3, in the experiment nº 1 – “Run 2” ...................................................................... 30
Table 13 – Results from hydrogen production batch tests, for the experiment nº 2 – “Run 1” .. 33
Table 14 – Mathematical model parameters, obtained by the sampled from the experiment nº2 –
“Run 1”........................................................................................................................................ 33
Table 15 – Results from hydrogen production batch tests, for the experiment nº 2 – “Run 2” . 35
Table 16 – Mathematical model parameters, obtained by the sampled from the experiment nº2 –
“Run 2”........................................................................................................................................ 35
Table 17 – The pure bacterial cultures for fermentative hydrogen production .......................... 33
iv
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
ABBREVIATIONS AND ACRONYMS
BHP Biological Hydrogen Production
BMP Biological Methane Potential
DNS Dinitrosalicylic
ERASMUS European Region Action Scheme for the Mobility of University Students
FID Flame Ionization Detector
HPM Hydrogen Production Medium
NB Nutrient Broth
NI Non Inoculated
OD Optical Density
TCD Thermal Condutivity Detector
TKN Total Kjeldahl Nitrogen
TOC Total Organic Carbon
TS Total Solids
UASB Upflow Anaerobic Sludge Blanket
VFA Volatile Fatty Acids
VS Volatile Solids
v
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
PREFACE
Since I decided to enroll in the Porto University, one of my main goals was to know if I
would be able to study in a different country and in a completely different environment
than the one I am used to. The best moment happened in the last year of the Master in
Environmental Engineering, at the completion of the dissertation.
In this sense, the ERASMUS programme was the opportunity for which I was
expecting. This programme provided me an extremely enriching academic and personal
experiment. Facing new reality encompasses challenges that tend to be overcome with
our larger involvement in a different society. These are constant challenges which
promote this international experiment. The effort to achieve the academic and personal
goals makes it unique and thus, it is possible to develop single qualities such as
autonomy, responsibility and curiosity for the “know more”.
As final stage of the course, I would like to work on the topic about solid waste
treatment, because it is a subject that always captured my interest and nowadays it is a
field with numerous researches, which will lead to the possibility of improving the
planet future, for example, in the field of sustainable energy production. Furthermore, I
have always had the idea to perform my thesis in an experimental research or apply my
knowledge on a work in the field.
For all these reasons, the ERASMUS programme complemented my academic path to
enable the realization of my thesis at the Department of Civil, Environmental and
Architectural Engineering of the University of Padova, in Italy. With the cooperation of
Prof. Raffaello Cossu and Dr. Luca Alibardi, I had the opportunity to conduct my thesis
in the Laboratory of Environmental and Sanitary Engineering of the University of
Padova that is located in Voltabarozzo, a quartier in the south part of Padova. Dr. Luca
Alibardi, my co-supervisor, proposed to me to work on biological hydrogen production
(BHP) tests and so, the theme of my thesis is the Biological Hydrogen Production using
organic waste and specific bacterial species.
In March of 2013 my research began with the following main objectives: evaluate the
biological hydrogen production potentials from a real mixed bacteria culture, using
granular sludge samples collected at different times from a real scale plant; evaluate the
biological hydrogen production potentials from pure cultures of single bacteria species;
vi
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
comparison between a mixed and a pure culture of bacteria, in terms their maximum
hydrogen productions and rates; evaluate the behavior of the best mixed culture in
different food/microorganisms ratio conditions.
With all the work that I performed in the laboratory I acquired skills on the lab scale
procedure for the evaluation of biological hydrogen production potentials by
fermentation processes and I also worked in the biological methane potential (BMP)
tests, in order to understand the behavior that each sample had at the second stage of the
anaerobic digestion process. This last subject will not be analyzed in this work, since
the main objective is the biological hydrogen production (BHP), but the results of BMP
tests can be consulted in Annex 1. Moreover I acquired competences and autonomy on
physical and chemical analysis of organic materials, such as: Total Solids (TS), Volatile
Solids (VS), Total Organic Carbon (TOC), Total Kjeldahl Nitrogen (TKN), Ammonium
nitrogen and Total Phosphorous, Chemical Oxygen Demand (COD), Optical Density
(OD), Titration at 7 points, Volatile Fatty Acids (VFA), water displacement method and
qualitative biogas measurements by gascromatographic techniques.
Before starting the experimentation on the two stage anaerobic digestion process, it was
necessary to decide the working conditions: the F/M ratio, the concentrations of
substrate and inoculum to use, and the pH at which the tests would have been
conducted. With this purpose I started my literature research on the web and on
scientific articles, but I also read the previous theses done in the laboratory by other
students, to understand what I could do to improve the experiment or to avoid mistakes
already done. Actually the parameters were changing in every experiment, because of
the great variability that substrates were displacing, or because of the different systems
that were used. Sometimes it was also difficult to find a comparison between different
works.
Despite all the difficulties that appeared, such as finding a house to live in, move by
bicycle with rain and wind, a different language to learn and mainly be far away from
my family and friends, this experiment abroad helped me not only to expand my
knowledge on a theme that is nowadays as concrete as unknown, also to face the lab
approach and to deal with a real research and most important to realize that I am able to
overcome the most difficult challenges in my life.
vii
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
1. INTRODUCTION
Hydrogen is a valuable gas that is currently used as feedstock for in several industries
processes. Hydrogen is also considered to be a clean energy source. Therefore, demand
on hydrogen production has increased considerably in recent years. Electrolysis of
water, steam reforming of hydrocarbons and auto-thermal processes are well-known
methods for hydrogen gas production, but not environmentally sustainable due to high
energy requirements, mainly based on fossil fuels. In this sense, biological production
of hydrogen gas has significant advantages over chemical methods (Kapdan & Kargi,
2006). In fact, this is an exciting scientific area since it is dealing with the conversion of
low costs residues or organic waste to a valuable energetic source, hydrogen (Hu et al.,
2013).
Biological hydrogen production from renewable sources has received considerable
attention in recent years. The biological processes utilized for hydrogen gas production
mainly are bio-photolysis of water by algae or cyanobacteria, dark fermentation and
photo fermentation of organic materials. The process of hydrogen production by
fermentative bacteria, not light dependent, is known as dark fermentation and takes
place during the fermentative or acidogenic phase of anaerobic digestion. Dark and
photo fermentation processes are considered more environmental beneficial and feasible
due to simultaneous waste treatment and hydrogen production. However, dark
fermentation is faster than photo fermentation and it has several advantages like no light
dependency, high production rates and efficiency, it can use various organic waste and
wastewater enriched with carbohydrates (Hu et al., 2013). On the other hand, dark
fermentation represents not only an energy production process but also a first stage of
stabilization for organic substrates since it degrades complex organic matter to readily
biodegradable compounds (volatile fatty acids and alcohols) suitable for methane
production by anaerobic digestion (Kapdan & Kargi, 2006).
Anaerobic treatment of complex organic materials is normally considered to be a two-
stage process. In the first stage, the complex organics are changed in form by a group of
facultative and anaerobic bacteria commonly termed the “acid formers”. Complex
materials such as fats, proteins and carbohydrates are hydrolyzed, fermented and
biologically converted to simple organic materials. For the most part, the end products
of this first-stage conversion are organic fatty acids (McCarty, 1964).
1
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Although no waste stabilization occurs during the first stage of treatment, it is required
to place the organic matter in a form suitable for the second stage of treatment. It is in
the second stage of methane fermentation that real waste stabilization occurs. During
this stage, the organic acids are converted by a special group of bacteria termed the
“methane formers” into the gaseous end products, carbon dioxide and methane. The
largest percentage of methane will still result from acetic acid fermentation, which is the
most prevalent volatile acid produced by fermentation of carbohydrates, proteins and
fats. Acetic and propionic acid, on the other hand, are formed mainly during
fermentation of carbohydrates and proteins. The other volatile acids, although
significant are of minor importance (McCarty, 1964).
The purpose of biological hydrogen studies is to develop commercially practical
hydrogen production processes by exploiting hydrogen producing ability of
microorganisms through modern biotechnology (Debabrata & Nejat, 2001). Due to the
fact that solar radiation is not a requirement, hydrogen production by dark fermentation
does not demand much land and is not affected by the weather condition. Hence, the
feasibility of the technology yields a growing commercial value. Biological dark
fermentation is also a promising hydrogen production method for commercial use in the
future. With further development of these technologies, biomass will play an important
role in the development of sustainable hydrogen economy (Ni et al., 2006).
In this perspective, this study was performed, via dark fermentation, with the following
main objectives: i) evaluate the biological hydrogen production potentials from a real
mixed bacteria culture, using granular sludge samples collected at different times from a
real scale plant; ii) evaluate the biological hydrogen production potentials from pure
cultures of single bacteria species; iii) comparison between a mixed and a pure culture
of bacteria, in terms their maximum hydrogen productions and rates; iv) evaluate the
behavior of the best mixed culture in different food/microorganisms ratio conditions.
To maximize hydrogen production via dark fermentation, methanogens and hydrogen-
consuming bacteria should be inhibited. Moreover, optimal process conditions, as type
and pre-treatment of inoculum, pH, temperature and substrate characteristics, should be
defined in order to promote the metabolic pathways resulting in hydrogen production
(Fang & Liu, 2002).
2
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Anaerobic sludges collected from full-scale digesters are frequently reported to be used
as inocula for hydrogen production and their pre-treatments are essential for the
inhibition of methanogenic microbial species (Van Ginkel & Sung, 2001; Ting et al.,
2004; Tommasi et al., 2008). Several methods have been proposed to achieve this aim,
including the heat treatment that is widely applied due to its effectiveness on
methanogenic inhibition (Alibardi et al., 2009).
Therefore, in this study, the seed material used for the evaluation of the biological
hydrogen production from a real mixed bacteria culture was a granular sludge collected
from a real scale Upflow Anaerobic Sludge Blanket (UASB) anaerobic digester of a
brewery factory located in Padova, Italy. This granular sludge used was pre-treated,
based on a heating procedure, prior to starting the experimental tests as inocula of batch
test for hydrogen production.
For organic materials to be potentially useful as substrates for sustainable biohydrogen
production, they must be not only abundant and readily available but, also, cheap and
highly biodegradable (Guo et al., 2010). Glucose and sucrose are the fermentation
substrates most studied in the laboratory. Thus, glucose was the substrate used as carbon
source, since it is a common and abundant substrate that could come from hydrolysis of
starch or cellulosic feedstock. Glucose is also shown to be a very effective substrate for
fermentative hydrogen production, leading to excellent hydrogen productivities.
Some species of indigenous microbial population of organic waste may have good
characteristics for the hydrolysis of complex substrates into simple monomers and for
an efficient conversion into hydrogen. In this perspective, organic waste could serve not
only as a substrate for hydrogen production but also as a source of hydrogen producing
bacteria (Favaro et al., 2013). Therefore, the efficiency of energy production can be
improved by screening microbial diversity and easily fermentable feed materials (Kalia
& Purohit, 2008).
Among the dark fermentative hydrogen producers pure cultures known to produce
hydrogen from carbohydrates include species of Enterobacter, Bacillus and
Clostridium. The latter two groups are characterized by the formation of spores in
response to unfavorable environmental conditions such as lack of nutrients or rising
temperature (Hawkes et al., 2002).
3
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
In 2011 an investigation was conducted at the Laboratory of Environmental and
Sanitary Engineering of the University of Padova which aimed at the development of an
efficient microbial inoculum for the industrial conversion of organic residues into
hydrogen. The presence of different extracellular enzymatic activities in many Bacillus
sp. should be considered promising towards the definition of a proper inoculum for the
conversion of complex organic wastes into hydrogen (Favaro et al., 2011). Bacillus
genus shows many features appropriate for hydrogen production: they can survive under
harsh conditions, hence could compete with other microbes; they have large and
versatile enzymatic activities such as lipase, amylase, protease and cellulose, hence a
diverse range of bio-wastes could be used as substrate for bio-hydrogen production;
they do not require light for hydrogen production; Bacillus spores are being used as
probiotics in humans and animals, thus they may not pose environmental concerns
(Kalia & Purohit, 2008).
In literature, indeed, Bacillus sp. is considered as a strong candidate for biological H2 –
production, because of its unique traits. For this purpose, Bacillus licheniformis,
Paenibacillus cookie, Bacillus sp., Paenibacillus sp. and Bacillus farraginis were
investigated and compared at different glucose concentrations. In addition, relationship
between different substrates used, which were glucose and Nutrient Broth (NB), and
cumulative hydrogen productions were also evaluated.
Although the microbiology and biochemistry of the anaerobic process is complex, it
normally operates quite well with a minimum control. The bacteria responsible for this
treatment are widespread in nature and grow well by themselves when provided with a
proper environment (McCarty, 1964).
The results from this study were expected to be helpful for understanding the behavior
of anaerobic hydrogen production process, using pure and mixed anaerobic bacteria
cultures in batch mode.
4
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
2. EXPERIMENTAL PROCEDURES
2.1 Research Scheme
The present research work was divided into three fundamental parts related respectively
with the proposed objectives, which are concretely:
Part 1 – Biological hydrogen production from a real mixed bacteria culture;
Part 2 – Experiments in a batch mixed reactor.
Part 3 – Biological hydrogen production potentials from pure cultures of single
bacteria species;
All the experiments were carried out at batch level and the results were assessed using
the experimental data and mathematical models applied to cumulative hydrogen
productions.
2.2 Inoculum conditioning and characterization
2.2.1 Real mixed bacteria culture
The seed material used for the evaluation of the biological hydrogen production from a
real mixed bacteria culture, was a granular sludge collected from a real scale Upflow
Anaerobic Sludge Blanket (UASB) anaerobic digester of a brewery factory located in
Padova, Italy.
In this experimental work three specific types of granular sludge were studied. The three
samples have been identified as samples “Sludge 2011”, “Sludge 2012” and “Sludge
2013”, which differ in the year of collection, that were 2011, 2012 and 2013,
respectively.
The granular sludge used was pre-treated prior to starting the experimental tests as
inocula of batch test for H2 production. This pre-treatment is based on a heating
procedure consisting of boiling the sludge sample at a fixed temperature of 100° C for 4
hours, in an oven.
5
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
This heat treatment of inoculum was evaluated to be optimal for selecting H2 producing
microorganisms, characterized by high H2 conversion yields, and for inhibiting of the
methanogenic activity (Alibardi et al., 2012).
The three samples of the granular sludge used for hydrogen production tests were
characterized by analyzing the content of: Total Solids (TS), Volatile Solids (VS), Total
Organic Carbon (TOC), Total Kjeldahl Nitrogen (TKN), Ammonium nitrogen and Total
Phosphorous, and these results are reported in Table 1. It is important to note that heat
treatment did not change the physical-chemical composition of the sludge.
Table 1. Physical and chemical characterization of the granular sludge samples.
Parameter Sample Name
Sludge 2011 Sludge 2012 Sludge 2013
TS±SD [%] 9 ± 1 13 ± 1 10 ± 1
VS±SD [% of TS] 73 ± 1 72 ± 1 80 ± 1
TOC±SD [% of TS] 39 ± 1 40 ± 1 45 ± 1
COD±SD [mgCOD/gTS] 1164 ± 5 1248 ± 5 1273 ± 5
TKN±SD [mg-N/g-TS] 67.6 ± 0.5 77.9 ± 0.5 82.0 ± 0.5
NH4+±SD
[mg-N/g-TS] 29.2 ± 0.5 13.9 ± 0.5 20.2 ± 0.5
Ptot±SD [mg-P/g-TS] 19.6 ± 0.5 13.3 ± 0.5 18.0 ± 0.5
2.2.2 Batch mixed reactor
For the purpose to evaluate hydrogen production rates at different F/M ratios (food over
microorganism ratio) a system was created, working as a batch reactor. This system was
provided of a continuous pH monitoring and controlling system and a continuous biogas
production monitoring system.
The seed material used for the evaluation of the biological hydrogen production from a
real mixed bacteria culture, was the granular sludge from 2013 (the physical and
chemical characterization of this granular sludge is presented in Table 1). This sludge
was selected because it showed, in the first part of this work, the best performance in
terms of hydrogen production rate. As previously reported, the granular sludge used was
pre-treated prior to starting the experimental tests as inocula of batch test for H2
production.
6
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
2.2.3 Pure bacteria culture
In order to work on the utilization of pure cultures for the development of a specific
inoculum for hydrogen production from organic substrates, an internal co-operation was
established with colleagues from the Department of Agronomy Food Natural Resources
Animals and Environment of the University of Padova.
In 2011 an investigation was conducted which aimed at the development of an efficient
microbial inoculum for the industrial conversion of organic residues into hydrogen.
One hundred and twenty microbial strains, previously isolated from mixed consortia
with interesting H2 fermentative performances from glucose, were genetically identified
and screened for their extracellular hydrolytic profile on the main components of the
organic fraction of municipal solid waste (OFMSW). In the end few Bacillus sp. isolates
showed promising hydrolytic capabilities (Favaro et al., 2011).
In this sense, the bacteria species that showed more promising hydrolytic capabilities
were evaluated in this present work, as pure cultures, for their H2 production potentials
from both simple and complex substrates.
Therefore, will be analysed their main role as inocula of batch test for H2 production,
and then will be evaluated their activity compared with the real mixed bacteria culture
activity. The Table 2 shows the microbial strains that were used.
Table 2. Identification of the microbial strains used and their hydrolytic abilities.
Microbial strains used Microbial sample name Hydrolytic abilities
Bacillus licheniformis
LF1.33 A
Cellulose
Hemicellulose
Starch
Protein
Paenibacillus cookie
LF2.3 B Starch
Bacillus sp.
LF2.8 C
Starch
Protein
Paenibacillus sp.
LF4.8 D
Pectin
Protein
Bacillus farraginis
LF2.7 E
Starch
Protein
7
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Two specific experiments were defined in which the variables were the bacteria species
and the substrate used. Each experiment was divided in two sub-parts, named Run 1 and
Run 2. The delineation of both experiments is reported in Table 3.
Table 3. Delineation of the experimental activities performed in batch test.
Experiment performed Microbial
sample name
Substrate added
Composition Quantity [g/l]
Experiment Nº 1
Run 1
A Glucose
Yeast Extract
5
3
B Glucose
Yeast Extract
5
3
C Glucose
Yeast Extract
5
3
Run 2
A1, B1, C1 ---- 0
A2, B2, C2 Glucose 5
A3, B3, C3 Glucose 10
Experiment Nº 2
Run 1
A Nutrient Broth* 5
B Nutrient Broth* 5
C Nutrient Broth* 5
D Nutrient Broth* 5
E Nutrient Broth* 5
Run 2
A Glucose 5
B Glucose 5
C Glucose 5
D Glucose 5
E Glucose 5
*Nutrient Broth (NB) is composed by: peptone bacteriological (5 g/l); beef extract (1.5 g/l); yeast extract (1.5 g/l); NaCl (5.0 g/l).
8
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
2.3 Batch test for hydrogen production
2.3.1 Real mixed bacteria culture
The hydrogen production tests were performed in batch reactors under mesophilic
conditions. Batch reactors consist in 0.5 liter Pyrex vessels, hermetically closed by
means of a plug with a silicone septum that allows the gas and water sampling with a
syringe. The working volume of 250 ml of the reactor is made up of: the substrate,
which was glucose, C6H12O6, (at a concentration of 5 g/l); the seed material (50 g of
granular sludge) and the required phosphate buffer solution to set the pH at 5.5, which
was the optimum for hydrogen production by mixed anaerobic cultures obtained by
many previous studies (Van Ginkel & Sung, 2001; Fan et al., 2004). The ratio between
the volatile solids of the substrate to be degraded and the volatile solids of the inoculum
(food over microorganism ratio – F/M) was set at 0.35 gVS/gVS.
Anaerobic conditions were obtained by making nitrogen flow trough the head space of
the vessel for 3 minutes. After this operation the excess pressure was removed in order
to re-establish the atmospheric pressure. The mesophilic conditions were guaranteed by
keeping the reactors in a water bath at a steady temperature of 35° C (± 1° C).
Each test was carried in triplicate and were made two blank tests of each sample,
containing only the respective inoculum and the buffer, that were performed in order to
assess the biological hydrogen production of the sole biomass present into the sludge.
The amount of biogas produced was recorded daily, using the water displacement
method and biogas composition in terms of hydrogen, carbon dioxide and methane was
measured by a gas chromatograph, in this order quantity and the quality of the biogas
were measured. Liquid samples were collected at the end of the fermentation tests and
analyzed for the concentration of volatile fatty acids. These tests lasted 7 days and also
during this period the pH value of the digestion liquid was monitored.
9
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
2.3.2 Batch mixed reactor
The hydrogen production tests were performed in batch reactors under mesophilic
conditions that were integrated in a circuit between a pH control and monitoring system
and a bascule.
As the time that the biogas is formed it is directed to a channel that ends inside a wet tip
gas meter, which is inserted inside a container with water. Over time, the gas meter is
filled with a volume of biogas produced by the batch reactor. When 3.8 ml of biogas
remains inside the gas meter it moves and consequently the biogas is released out of the
system. This movement is shown automatically in the system and so, it is possible to
know at the end of the batch test the volume of biogas formed by the number of
movements performed by the wet tip gas meter.
Simultaneously there is a system for the pH control and monitoring, which makes
possible to maintain the more favorable pH value for the hydrogen production which is,
in this case, 5.5. This system is directly connected to the batch reactor by a pH meter,
which is connect to a device which triggers the introduction of sodium hydroxide
(NaOH), through a needle inserted into the batch reactor. Thus, whenever the pH value
decreases it is automatically introduced, in the reactor, a certain amount of NaOH to
achieve the pH value established as optimal.
For the purpose to evaluate hydrogen production rates at different F/M ratios (food over
microorganism ratio) three different batch tests were created. The following F/M ratios
were tested: 0.39 gVS/gVS; 0.78 gVS/gVS and 1.56 gVS/gVS. Each test were named as
“S_40”, “S_20” and “S_10” respectively. In these different tests were used batch
reactors for each of the four experiments, which consist in 0.5 liter Pyrex vessels,
hermetically closed by means of a plug with a silicone septum that allows the gas and
water sampling with a syringe. The reactor performance of each experiments was
assessed at four different quantities in granular sludge and so the working volume of
250 ml of the reactor is made up of: the substrate, which was glucose (C6H12O6) at 5 g/l
as concentration in all the four test; the seed material (granular sludge) with an amount
of 40 g, 20 g and 10 g, for each test respectively; distilled water and sulfuric acid
(H2SO4) with 0.1 M, to set the initial pH at 5.5.
10
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Anaerobic conditions were obtained by making nitrogen flow through the head space of
the vessel for 3 minutes. The mesophilic conditions were guaranteed by placing the
reactor in contact with a heating system programmed to keep the temperature at 35 °C
(± 1° C).
The amount of biogas produced, the pH variation and the NaOH added were recorded at
the end of each batch test, with the aid of a computer program, which is connected to
the batch system enabling the capture of a photograph, from 10 to 10 minutes, with the
information referred before.
When the fermentation test ends, the biogas composition in terms of hydrogen, carbon
dioxide and methane was measured by a gas chromatograph. Then a liquid sample was
collected and it was analyzed for the concentration of VFAs, Ammonium nitrogen and
Total Phosphorous and Titration at 7 points.
2.3.3 Pure bacteria culture
2.3.3.1 Experiment nº1
As previously reported this experimental phase was divided in two parts, which were
nominated as “Run 1” and “Run 2”.
In “Run 1” batch reactors, 0.5 liter Pyrex vessels were filled with 250 ml of hydrogen
production medium (HPM) containing glucose (5 g/l) and yeast extract (3 g/l) and the
required buffer solution, which was a phosphate buffer solution to set the pH at 5.5.
Each microbial strain was aerobically pre-grown in 200 mL Erlenmeyer flasks
containing 50 mL of HPM or Nutrient Broth (N.B) ( 2.5, 5, 7.5, 10 %, v/v). The growth
of each strain, inoculated at an initial optical density (OD 600nm) value of 0.06, was
monitored by determining the OD at 600 nm with a spectrophotometer (Ultrospec 2000,
Pharmacia Biotech).
After aseptically inoculated, the reactors were hermetically closed using a silicon plug.
Anaerobic conditions were obtained by making nitrogen flow trough the head space of
the vessel for 3 minutes. After this operation the excess pressure was removed in order
to re-establish the atmospheric pressure.
11
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
The mesophilic conditions were guaranteed by keeping the reactors in a water bath at a
steady temperature of 35° C (± 1° C).
Each test was carried in triplicate and was made one blank test, named as Non –
Inoculated (NI) sample, containing only the HPM solution and the buffer. The blank test
was performed in order to be sure that the sterilized conditions are maintained.
The amount of biogas produced was recorded daily, using the water displacement
method and biogas composition in terms of hydrogen, carbon dioxide and methane was
measured by a gas chromatograph, in this order the quantity and the quality of the
biogas were measured. Liquid samples were collected at the second day and at the end
of the fermentation tests and analyzed for the concentration of volatile fatty acids.
These tests took place over 5 days, after which no longer significant production of
hydrogen was noted. Also during this period the pH value of the digestion liquid and the
OD were monitored.
In the end of the “Run 1”, residual glucose in the HPM medium was measured
according to the dinitrosalicylic (DNS) method described by Miller (1959).
In “Run 2” was analyzed the behavior of each sample introducing different quality and
quantity of substrate. In this sense, it was done the following procedure: in samples
identified with the number 1 (A1, B1 and C1) the initial conditions were maintained and
so these samples were used as blank samples in “Run 2”; 5 g/l of glucose was added in
samples identified with the number 2 (A2, B2 and C2); 10 g/l of glucose was added in
samples identified with the number 3 (A3, B3 and C3).
The batch reactors, 0.5 liter Pyrex vessels, were filled with the amount of subtract
planed for each sample and the required buffer solution, which was a phosphate buffer
solution to set the pH at 5.5. After inoculation, all the procedure followed it is the same
as previously reported.
The amount of biogas produced was recorded daily, using the water displacement
method and biogas composition in terms of hydrogen, carbon dioxide and methane was
measured by a gas chromatograph, in this order the quantity and the quality of the
biogas were measured. Liquid samples were collected at the end of the fermentation
tests and analyzed for the concentration of volatile fatty acids.
12
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
These tests took place over 3 days, after which no longer significant production of
hydrogen was noted. Also during this period the pH value of the digestion liquid and the
optical density was monitored.
2.3.3.2 Experiment nº2
This experimental work was divided in two parts, which were nominated as “Run 1”
and “Run 2”.
In “Run 1” batch reactors, 0.5 liter Pyrex vessels, were filled with Nutrient Broth (NB)
and a phosphate buffer solution to set the pH at 5.5. After inoculation, all the procedure
followed was the same as previously reported. Each test was carried in triplicate and in
this experiment were made two blank tests, named as NB1 and NB2, containing only
the Nutrient Broth solution and the buffer. The blank test was performed in order to be
sure that the sterilized conditions are maintained.
The amount of biogas produced was recorded daily, using the water displacement
method. Biogas composition in terms of hydrogen, carbon dioxide and methane was
measured by a gas chromatograph, in this order the quantity and the quality of the
biogas were measured. Liquid samples were collected at the end of the fermentation
tests and analyzed for the concentration of volatile fatty acids. These tests took place
over 12 days, after which no longer significant production of hydrogen was noted. Also
during this period the pH value of the digestion liquid and the optical density was
monitored.
In “Run 2” it was analyzed the behavior of each sample introducing different quantity of
substrate. In this sense, it was added glucose (5 g/l) in all the samples. The batch
reactors, 0.5 liter Pyrex vessels, were filled with the amount of subtract planed for the
samples and the required buffer solution, which was a phosphate buffer solution to set
the pH at 5.5. After inoculation, all the procedure followed it is the same as previously
reported.
The amount of biogas produced was measured in exactly the same manner as the “Run
1”. Liquid samples were collected at the second day and at the end of the fermentation
tests and analyzed for the concentration of volatile fatty acids.
13
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
These tests took place over 7 days, after which no longer significant production of
hydrogen was noted. Also during this period the pH value of the digestion liquid and the
optical density was monitored.
2.4 Methods
2.4.1 Analytical methods
Total Solids (TS), Volatile Solids (VS), Total Kjeldahl Nitrogen (TKN), Ammonium
nitrogen and Total Phosphorous were analysed according to Standard Methods (APHA,
1999). Total Organic Carbon (TOC) was quantified using a Total Carbon Analyzer
(TOC – V CSN, Shimadzu).
Volatile Fatty Acids (VFAs) concentrations were analyzed using a gas chromatograph
(Varian 3800) equipped with flame ionization Detector (FID), Stabilwax – DA column,
nitrogen as carrier gas.
The composition of biogas in the headspace was measured by means using a micro-GC
(Varian 490-GC) equipped with a 10-meter MS5A column and a 10-meter PPU column.
Helium was used as carrier gas.
To analyze the Optical Density of the microorganisms inside the batch reactors it was
necessary collect 3 ml of each sample and measured it, using a spectrofotometer.
Subsequently the same samples were used to determine their pH, with the aid of a pH
meter.
2.4.2 Experimental data results
The amount of biogas produced by fermentation was measured by means of the
dislocation method. According to the functional principle of dislocation, the excessive
pressure formed into the head space of reactors moves a volume of liquid, present in
another bottle, equal to the volume of the biogas that was produced by fermentation.
The displaced liquid is an acid saline solution (pH ˂ 3 and 25% NaCl), where carbon
dioxide (CO2) and methane (CH4) do not dissolve into, and it is collected in a granular
cylinder.
14
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
The volume of hydrogen produced in the period of between two measurements, t and (t-
1), has been calculated according to the following equation (1) (Van Ginkel et al.,
2005):
where:
VC,t is the volume of H2 produced in the period of time between time t and time t-1;
CC,t is the concentration of hydrogen measured at time t;
VG,t is the volume of biogas produced in the period of time between time t and time t-1;
VH is the volume of the reactor headspace;
CC,t-1 is the concentration of hydrogen measured at time t-1.
2.4.3 Mathematical models of hydrogen production
To compare the results obtained from the batch tests, data were interpolated using a
Gompertz equation when dealing with a latency phase or a first order kinetics equation
in a situation of exponential production.
The Gompertz equation (2) used is as follows (Lay et al., 1997):
–
(2)
where:
B(t) is the cumulative biogas/hydrogen production at time t(d) (Nml/gVS);
B0 is the maximum biogas/hydrogen production (Nml/gVS);
R is the biogas/hydrogen production rate (Nml/gVS.d);
λ is the latency phase (d);
e is Euler’s number.
VC,t = CC,t VG,t + VH (CC,t – CC,t-1) (1)
15
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Average values of cumulative biogas and hydrogen production from each experimental
condition were used to obtain the values of the parameters B0, R and λ. These
parameters were estimated by minimizing the sum square of errors between
experimental data and results from the model. The estimations were carried out by using
the “Solver” function in Excel of Microsoft Office.
When no latency phase was detected, the data of hydrogen production were interpolated
using an exponential function (Trzcinski & Stuckey, 2012), as it is showed in the first
order kinetics equation bellow (3):
(3)
where:
P(t) is the cumulative biogas/hydrogen production at time t(d) (Nml/gVS);
P0 is the maximum biogas/hydrogen production (Nml/gVS);
k is the kinetics degradation constant (d-1
).
Average values of cumulative biogas and hydrogen production from each experimental
condition were used to obtain the values of the parameters P0 and k. These parameters
were estimated by minimizing the sum square of errors between experimental data and
results from the model. The estimations were carried out by using the “Solver” function
in Excel of Microsoft Office.
It is important to note that data of hydrogen yield are expressed as ml of hydrogen at
temperature of 0 °C and pressure of 1 atm.
16
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
3. RESULTS AND DISCUSSION
3.1 Biological hydrogen potential production
3.1.1 Real mixed bacteria culture
With the purpose to evaluate the biological hydrogen production potentials from a real
mixed bacteria culture, granular sludge samples, collected at different times from a real
scale plant, were used as inocula for this experiment.
The results of hydrogen production potentials are reported in Table 4. The mathematical
model parameters, obtained from the average cumulative hydrogen productions of the
experimental data, are described in Table 5 and the cumulative hydrogen productions
curves from experimental results and from mathematical models obtained by the
samples “Sludge 11”, “Sludge 12” and “Sludge 13” are showed in Figure 1. In the
Figure 2 it is possible to simultaneously compare the results obtained from the three
samples.
Table 4. Results from hydrogen production batch tests, obtained by each real mixed bacteria
culture sample.
Sample name
Hydrogen
yield±SD
[Nml H2 / gVS]
Volatile Fatty Acids
Acetic acid
[mg/l]
Propionic acid
[mg/l]
Butyric acid
[mg/l]
Isovaleric acid
[mg/l]
Sludge 2011 113.2 ± 20.5 221.3 40.2 45.3 35.6
Sludge 2012 119.6 ± 18.3 774.9 44.4 775.2 49.5
Sludge 2013 148.2 ± 1.2 806.1 46.7 807.2 112.3
Table 5. Mathematical model parameters, obtained by each real mixed bacteria culture sample.
Sample name
Exponential function parameters
P0 [Nml H2 / gVS] k [d-1
] Max rate
[(Nml H2 / gVS)*d-1
]
Sludge 2011 113.2 3.0 339.6
Sludge 2012 119.6 2.9 346.8
Sludge 2013 148.2 7.3 1081.9
17
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 8
Cu
mu
lati
ve h
yd
ro
gen
pro
du
cti
on
[N
ml
H2 /
g V
S]
Time [d] I Experimental data
Mathematical model
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 8
Cu
mu
lati
ve h
yd
ro
gen
pro
du
cti
on
[N
ml
H2 /
g V
S]
Time [d] II Experimental data
Mathematical model
0
20
40
60
80
100
120
140
160
180
0 1 2 3 4 5 6 7 8
Cu
mu
lati
ve h
yd
ro
gen
pro
du
cti
on
[N
ml
H2 /
g V
S]
Time [d] III Experimental data
Mathematical model
Figure 1. Cumulative hydrogen productions from average experimental data and from the
mathematical model by the samples: (I) “Sludge 2011”; (II) “Sludge 2012”; (III)
“Sludge 2013”. The vertical bars over the experimental data represent the standard
deviations of the triplicate.
18
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
20
40
60
80
100
120
140
160
0 2 4 6 8
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2/g
VS
]
Time [d]
Sludge 2011
Sludge 2012
Sludge 2013
Figure 2. Comparison between the cumulative hydrogen productions from the mathematical
model, by the samples “Sludge 2011”, “Sludge 2012” and “Sludge 2013”.
Methane was not detected, because of elimination of methane producers by heat
digestion of sludge. The highest hydrogen production was measured for the sample
“Sludge 2013”, with a total hydrogen production of 148.2 NmlH2/gVS, whereas the
sample “Sludge 2011” showed the lowest production, with a total hydrogen production
of 113.2 NmlH2/gVS.
According to the results, it is possible to conclude that the age of the sludge is directly
associated to the behavior of the bacteria, that are contained in the inoculum, and so, as
more recent is the granular sludge, higher hydrogen production will be achieved and
higher will also be the rate process.
The operational pH value is considered to be a crucial parameter during the
fermentation process, because it affects the hydrogenase activity and the metabolism
pathway (Vijayaraghavan & Soom, 2004). During the experimental activities the pH of
all the samples remained practically constant, ranging from 5.5 and 6.0, and
subsequently this parameter contributed to the achievement of high yields of hydrogen.
In order to identify the constraints of the dark fermentation process on the hydrogen
production, further analyses were done on the liquid samples to know the VFAs which
were formed during the fermentation process.
19
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
When bacteria metabolize simple sugars, such as glucose, organic acids are produced as
sub-products, since there is an incomplete conversion of the substrate to carbon dioxide
(Van Ginkel et al., 2005). The following equations show the production of these small
organic acids in two different ways. In one way, glucose in biomass gives a maximum
yield of 4 H2 per glucose when acetic acid is the by-product (Hawkes et al., 2002):
C6H12O6 + 2 H2O → 2 CH3COOH + 2 CO2 + 4 H2 (4 ATP) (3)
In other way, half of this yield per glucose is obtained with butyrate as the fermentation
end product (Hawkes et al., 2002):
C6H12O6 → CH3CH2CH2COOH + 2 CO2 + 2 H2 (3ATP) (4)
Until now it was not known why one way is favored over another, however the
combination of both is observed in all bacteria populations (Van Ginkel et al., 2005).
It is important to note that the overall equation for the production of propionate from
glucose, shows that this involves the consumption of H2 (Vavilin et al., 1995):
C6H12O6 + 2 H2 → 2 CH3CH2COOH + 2 H2O (5)
Thus the production of propionate should be avoided. Vavilin et al. (1995) stated that
the limiting substrate for butyrate production is glucose, while the limiting substrate for
propionate production is H2, and the two groups of organisms producing these end
products are in balance in the microbial consortium producing H2. Limiting the amount
of propionate-formers by heat treatment of the inoculum may aid in biasing the
community towards butyrate production.
Figure 3 presents the VFA composition formed by the bacteria during the fermentation
process.
20
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
100
200
300
400
500
600
700
800
900
Sludge 2011 Sludge 2012 Sludge2013
VF
A p
ro
du
cti
on
[ m
g/l
]
Acetic acid
Butyric acid
Propionic acid
Figure 3. Volatile fatty acids composition of dark fermentation the samples “Sludge 2011”,
“Sludge 2012” and “Sludge 2013”.
The previous results show that “Sludge 2013” had the higher acetate concentration
compared to “Sludge 2011” and “Sludge 2012”. These results agreed with the data on
hydrogen production.
It is also important to note that batch tests were conducted over 6 days during which
there was no addition of substrate and the initial conditions, which were the optimal,
tend to be altered. Such factors have as consequence an ecological bacteria selection due
to the limitation of “food” at “unfavorable conditions” and so a sharp reduction in the
number of bacteria occurs.
Furthermore, no lag phase was registered and consequently the biomass did not suffer
inhibition with the imposed substrate concentration and its adaption was not necessary
in this conditions (Maintinguer et al., 2008).
After the peak value of hydrogen production a phase of hydrogen consumption was
noted. These results can be seen in the Figure 4. In the Figure 5 it is possible to
simultaneously compare these results obtained from the three samples. Furthermore,
Table 6 shows the consumption rate of each sample.
21
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7
Cu
mu
lati
ve H
yd
ro
gen
Co
nsu
mp
tio
n
[Nm
lH2
/ g
VS
]
Time [d] I Experimental data
Trend line
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 Cu
mu
lati
ve
Hy
dro
gen
Con
sum
pti
on
[Nm
lH2 / g
VS
]
Time [d] II Experimental data
Trend line
0
30
60
90
120
150
180
0 1 2 3 4 5 6 7
Cu
mu
lati
ve H
yd
ro
gen
Co
nsu
mp
tio
n
[Nm
lH2 /
g V
S]
Time [d] III Experimental data
Trend line
0
30
60
90
120
150
180
0 1 2 3 4 5 6 7
Cu
mu
lati
ve
Hy
dro
gen
Con
sum
pti
on
s
[Nm
lH2 /
g V
S]
Time [d]
Experimental data of the Sludge 2011 Experimental data of the Sludge 2012
Experimental data of the Sludge 2013 Trend line, Sludge 2011
Trend line, Sludge 2012 Trend line, Sludge 2013
Figure 4. Cumulative hydrogen consumption from average experimental data by the samples:
(I) “Sludge 2011”; (II) “Sludge 2012”; (III) “Sludge 2013”. The vertical bars over the
experimental data represent the standard deviations of the triplicate.
Figure 5. Comparison between the cumulative hydrogen consumptions, by the samples “Sludge
2011”, “Sludge 2012 and “Sludge 2013”, and their trend lines.
22
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Table 6. Results from hydrogen consumption batch tests, obtained by each real mixed bacteria
culture sample.
Sample name Hydrogen consumption ± SD
[Nml H2 / gVS] Time [d]
Rate
[(Nml H2) / (gVS*d)]
Sludge 2011 31.6 ± 10.7 5.0 6.3
Sludge 2012 38.9 ± 10.7 4.0 9.7
Sludge 2013 27.6 ± 3.7 5.0 5.5
The previously graphs allows to understand that sample “Sludge 2013” had the lower
consumption rate, which was 5.5 Nml H2/(gVS*d) , in turn the sample “Sludge 2012”
showed the highest consumption rate equal to 9.7 Nml H2/(gVS*d).
These results mean that the sample “Sludge 2013”, which is the most recent, provides
the best global conditions since it shows the highest hydrogen production, as well as the
lowest rate of hydrogen consumption.
The hydrogen consumption is a phenomenon that presumably occurs because
homoacetigenic bacteria consume hydrogen to produce acetic acid and hydrogenases
bacteria recycle a portion of hydrogen produced (Hellenbeck & Benemann, 2002).
Homoacetogenic bacteria are strictly anaerobic microorganisms which catalyze the
formation of acetate from H2 and CO2. Unfortunately, the pretreatment of the inoculum
by heating to select spore-forming bacteria is not suitable for inhibiting of
homoacetogenic bacteria (Guo et al., 2010).
Thus, the reasons that lead to a better performance of the sample nominated as “Sludge
2013”, comparatively to the other samples, are: efficacy of the heat treatment, by
selecting H2 producing microorganisms and inhibiting methanogenic activity; higher
number of fermentative bacteria after the heat-treatment of the inoculum and lower
number in hydrogen consumers microorganisms, as homoacetogenic bacteria, that
compose the inoculum.
23
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
3.1.2 Batch mixed reactor
For the purpose to evaluate hydrogen production rates at different F/M ratios (food over
microorganism ratio) a system was created, working as a batch reactor. In this sense,
three different batch tests, which were named as “S_40”, “S_20” and “S_10”, were
studied. The results of the three tests can be seen in the Figure 6. Moreover, Table 7
shows the detailed results of the experimental data, using Gompertz equation as
mathematical model and in Table 8 are represented the analytical analyses, by each
sample.
Figure 6. Comparison between the hydrogen production rates, by the samples “S_40”, “S_20”
and “S_10”.
Table 7. Results from hydrogen yields and rates at different F/M ratios, from the mathematical
model, by each sample using the batch mixed reactor.
Sample name Inoculum added
[g]
Hydrogen yields
[Nml H2 / gVS]
Rate
[(Nml H2) / (gVS*d)] λ
[d]
S_40 40.0 88.9 6.3 0.39
S_20 20.0 91.2 9.7 0.37
S_10 10.0 77.5 5.5 0.28
0
10
20
30
40
50
60
70
80
90
100
0 3 6 9 12 15 18 21 24
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [h]
S_40
S_20
S_10
24
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Table 8. Results from analytical analysis, by each sample using the batch mixed reactor.
Sample
name
Ammonium
Nitrogen
[mgN/l]
Total
Phosphorous
[mgP/l]
Total
Alcalinity
[mg/l as
CaCO3]
Volatile Fatty Acids
Acetic
acid
[mg/l]
Propionic
acid
[mg/l]
Butyric
acid
[mg/l]
Isovaleric
acid
[mg/l]
S_40 161.0 107.7 5993.4 276.0 21.0 257.0 ˂ 10
S_20 68.6 53.6 2289.1 226.0 ˂ 10 173.0 ˂ 10
S_10 16.8 21.0 1653.9 172.0 ˂ 10 65.3 ˂ 10
The main objective of this experiment is to verify the relation between the amount of
inoculum added and its rate of hydrogen production, as well the hydrogen yields and so
the results allow to understand that:
The behavior of each sample, in terms of analytical analysis, is similar taking
into account the amount of inoculum added in each test, as the results in Table 8
show;
The hydrogen produced is independent of the amount of inoculum added, since
all tests achieved approximately the same amount of hydrogen yields;
On the contrary, the quantity of inoculum added is responsible on the larger or
smaller duration of the lag phase, as well as the rate of hydrogen production.
Since all the tests showed different performances with regard to these
parameters.
3.1.3 Pure bacteria culture
Enhancing the hydrogen production efficiency is one of the major challenges to dark
hydrogen fermentation. To achieve such a purpose, numerous research studies on
anaerobic microbes have been intensively developed in recent years, and some new or
efficient bacterial species and strains for dark hydrogen fermentation have been isolated
and recognized (Fang et al., 2002).
Therefore, the research direction is to improving both hydrogen yield and hydrogen
production rate simultaneously. In order to yield as much hydrogen as possible, it has to
be created one optimal microbial metabolism.
25
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
In this sense, this part of the study will investigate the hydrogen production potential of
five dark fermentative bacteria (Bacillus licheniformis, Paenibacillus cookie, Bacillus
sp., Paenibacillus sp., Bacillus farraginis) using glucose as substrate, available in
different concentrations, under anaerobic conditions. Will also be compared the
hydrogen production potential using Nutrient Broth (NB) as substrate.
3.1.3.1 Experiment nº 1: “Run 1”
In this experiment glucose was used as substrate, with a concentration of 5 g/l, in each
sample. The results of hydrogen production potentials are reported in Table 9. Even
more the mathematical model parameters, obtained from the average cumulative
hydrogen productions of the experimental data, are described in Table 10.
The cumulative hydrogen productions curves, from mathematical models, and the OD
and pH values obtained by the samples A, B and C are showed in Figure 7.
Table 9. Results from hydrogen production batch tests, for the experiment nº1–“Run 1”.
Sample
name
Hydrogen yield±SD
[Nml H2 / gVS]
Volatile Fatty Acids
Acetic acid
[mg/l]
Propionic acid
[mg/l]
Butyric acid
[mg/l]
Isovaleric acid
[mg/l]
A 9.5 ± 2.0 103.6 8.4 1.0 1.9
B 40.7 ± 30.2 91.5 3.1 8.4 3.1
C 10.8 ± 2.6 111.8 7.8 1.52 5.3
Table 10. Mathematical model parameters, obtained by the sampled from the experiment nº1 –
“Run 1”.
Sample
name
Parameters of the Gompertz equation
B0 [Nml H2 / gVS] λ [d] R
[(Nml H2) / (gVS*d)]
A 9.6 2.7 9.9
B 41.9 1.9 23.3
C 10.8 2.2 14.0
26
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5 6
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
g V
S]
Time [d] B Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
lH2/g
VS
]
Time [d] C Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] A Experimental data
Mathematical model
Optical Density
pH
Figure 7. Optical Density and pH variations, over time, and cumulative hydrogen productions
from average experimental data and from the mathematical model by the samples: (A)
B. licheniformis; (B) P. cookie; (C) Bacillus sp. The vertical bars over the
experimental data represent the standard deviations of the triplicate.
27
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
The highest hydrogen production was measured for P. cookie (sample B), with a total
hydrogen production of 41.9 NmlH2/gVS, in turn. Bacillus sp. (sample C) showed a
total hydrogen production of 10.8 NmlH2/gVS, whereas B. licheniformis (sample A)
showed the lowest production, with a total hydrogen production of 9.6 NmlH2/gVS.
The previously graphs show that the behavior of B. licheniformis and of Bacillus sp. are
very similar. However P. cookie shows a distinct behavior compared to the other two
samples.
The species B. licheniformis and Bacillus sp. show initially a clear rise in the microbial
density accompanied by a decrease of pH. Approximately after 1 day is evident an
evolution in the volume of fermentative bacteria present in the samples, corresponding
to a maximum value of 1.014 in sample B. licheniformis , and a maximum value of
0.985 in sample Bacillus sp.
Regarding specie P. cookie, this shows a more gradual behavior, in respect of the
increase of the number of fermentative bacteria and the decrease of the pH. A maximum
value of 1.212 was obtained, practically at the end of 5 days. However, in this case, the
maximum OD value obtained is practically the same as in B. licheniformis and in
Bacillus sp.
Actually, carbohydrates are the preferred organic carbon source for hydrogen-producing
fermentations (Hawkes et al., 2002). Thus, and according to the obtained results, it is
apparent that microbial species present in each sample consume the glucose, previously
added, leading to an increase in OD value. When the carbon source is no longer
available, the OD value decreases, remaining constant thereafter.
Furthermore, it is noted an existence of a lag phase in all the samples. This means that
the biomass suffered inhibition with the imposed substrate concentration and
consequently a period of adaptation to new environmental conditions occurred. In fact,
the lag phase can be related to the hydrogen productions, since P. cookie showed a lag
phase with the lowest period and the highest hydrogen production, contrary to what
occurred with B. licheniformis.
Initial pH influences the extent of lag phase in batch hydrogen production. Composition
of the substrate, media composition, temperature and the type of microbial culture are as
well important parameters affecting the duration of lag phase (Kapdan & Kargi, 2006).
28
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
As previously mentioned, it is possible to observe that there was a variation of the pH in
all the samples. In fact, it decreases evidently during the first phase for B. licheniformis
and Bacillus sp., and in opposite, for P. cookie, it decreases in the exponential phase of
the hydrogen production. In this sense, can be stated that the decrease in its value, due
to production of organic acids, depletes the buffering capacity of the medium resulting
in a lower final pH (Patel et al., 2012).
3.1.3.2 Experiment nº 1: “Run 2”
In this experiment glucose was used as substrate, with a concentration of 5 g/l (in
samples A2, B2 and C2) and a concentration of 10 g/l (in samples A3, B3 and C3). The
results of hydrogen production potentials are reported in Table 11. Even more the
mathematical model parameters, obtained from the average cumulative hydrogen
productions of the experimental data, are described in Table 12.
The cumulative hydrogen productions curves, from mathematical models, and the OD
and pH values obtained by the samples A2 and A3, B2 and B2 and C2 and C3 are
showed in Figure 8, 9 and 10, respectively. In the Figure 11 it is possible to
simultaneously compare the results obtained from all the samples with different quantity
in substrate.
Table 11. Results from hydrogen production batch tests, for the samples A2, B2 and C2 and for
the samples A3, B3 and C3, in the experiment nº 1 – “Run 2”.
Sample
name
Hydrogen yield
[Nml H2 / gVS]
Volatile Fatty Acids
Acetic acid
[mg/l]
Propionic acid
[mg/l]
Butyric acid
[mg/l]
Isovaleric acid
[mg/l]
A2 14.8 209.8 4.4 6.6 3.2
B2 27.9 129.3 2.1 5.4 3.7
C2 24.0 69.9 7.8 105.3 4.6
A3 2.7 97.0 11.2 11.5 1.6
B3 29.4 55.5 4.1 1.0 3.0
C3 9.9 199.8 7.5 6.3 5.4
29
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
1
2
3
0 1 2 3
Op
tica
l De
nsi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
g V
S]
Time [d] A3 Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
14
16
0 1 2 3
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
g V
S]
Time [d] A2 Experimental data
Mathematical model
Optical Density
pH
Table 12. Mathematical model parameters, for the sample A2, B2 and C2 and for the samples
A3, B3 and C3, in the experiment nº 1 – “Run 2”.
Sample
name
Parameters of the Gompertz equation
B0 [Nml H2 / gVS] λ [d] R
[(Nml H2) / (gVS*d)]
A2 15.1 0.2 23.3
B2 26.8 0.1 50.6
C2 24.6 0.2 20.2
A3 2.7 0.3 2.9
B3 29.5 0.4 70.7
C3 9.7 0.2 15.6
Figure 8. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A2 and A3.
30
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
5
10
15
20
25
30
0 1 2 3
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] B2 Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
5
10
15
20
25
30
35
0 1 2 3
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] B3 Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
5
10
15
20
25
30
0 1 2 3
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] C2 Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
0 1 2 3
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] C3 Experimental data
Mathematical model
Optical Density
pH
Figure 9. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples B2 and B3.
Figure 10. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples C2 and C3.
31
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
5
10
15
20
25
30
0 1 2 3
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] I Sample A2
Sample B2
Sample C2
0
5
10
15
20
25
30
35
0 1 2 3
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n [
Nm
l H
2 /
gV
S]
Time [d] II Sample A3
Sample B3
Sample C3
Figure 11. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A2, B2 and C2 and by the samples A3, B3 and C3,
with: (I) 5 g/l of glucose; (II) 10 g/l of glucose.
It is possible to observe that during the early stage, without lag phase, the initial glucose
concentrations had less impact on hydrogen production and so cellular growth
increased. After this period, the hydrogen production increased rapidly, until reaching a
maximum value of hydrogen production, from which the production practically ceased.
During the exponential phase of hydrogen production, pH was gradually decreased.
In this sense, the main differences between the samples examined lies in the maximum
production rate, given by the parameter R in the Gompertz equation, and also in the
maximum production yield, (see Table 12).
It was observed that at low glucose concentration (5 g/l), the cumulative hydrogen
production was 26.8 NmlH2/gVS for P. cookie (sample B2), while B. licheniformis
(sample A2) produced 15.1 NmlH2/gVS.
By increasing the glucose concentration to double (10 g/l), the maximum hydrogen
production was observed, as before, for P. cookie (sample B3), with a production of
29.5 NmlH2/gVS (value quite similar to the previously result). On the contrary, the
species B. licheniformis (sample A3) and Bacillus sp. (sample C3) followed opposite
32
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
tendency, showing a production of 2.7 NmlH2/gVS and a production of 9.7
NmlH2/gVS, respectively. These results are much lower than the results obtained with a
glucose concentration at 5 g/l.
Therefore, the maximum values of hydrogen production were recorded when the
glucose concentration was set to 5 g/l, suggesting that high glucose concentration could
inhibit the fermentation process.
3.1.3.3 Experiment nº 2: “Run 1”
In this experiment Nutrient Broth (NB) was used as substrate, with a concentration of 5
g/l, in each sample. NB is composed by: peptone bacteriological (5 g/l); beef extract
(1.5 g/l); yeast extract (1.5 g/l); NaCl (5.0 g/l).
The results of hydrogen production potentials are reported in Table 13. Even more the
mathematical model parameters, obtained from the average cumulative hydrogen
productions of the experimental data, are described in Table 14.
The cumulative hydrogen productions curves, from mathematical models, and the OD
and pH values obtained by the samples A, B, C, D and E are showed in Figure 12.
Table 13. Results from hydrogen production batch tests, for the experiment nº 2 – “Run 1”.
Sample
name
Hydrogen yield±SD
[Nml H2 / gVS]
Volatile Fatty Acids
Acetic acid
[mg/l]
Propionic acid
[mg/l]
Butyric acid
[mg/l]
Isovaleric acid
[mg/l]
A 8.3 ± 0.4 99.0 7.1 2.8 23.0
B 11.9 ± 0.4 81.5 9.3 2.4 14.2
C 11.1 ± 0.5 85.6 6.0 2.6 8.4
D 9.6 ± 1.1 64.8 8.6 2.7 1.4
E 12.5 ± 3.6 80.1 9.5 4.6 4.1
Table 14. Mathematical model parameters, obtained by the sampled from the experiment nº2 –
“Run 1”.
Sample
name
Parameters of the Gompertz equation
B0 [Nml H2 / gVS] λ [d] R
[(Nml H2) / (gVS*d)]
A 8.3 1.6 4.0
B 12.3 4.5 6.2
C 11.3 4.1 5.0
D 9.9 4.3 5.9
E 11.9 1.1 8.3
33
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0,000
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
lH2 / g
VS
]
Time [d] A Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
lH2/g
VS
]
Time [d] B Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d] C Experimental data
Mathematical model
Optical Density
pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0
2
4
6
8
10
12
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
gV
S]
Time [d] D Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
lH2 /
gV
S]
Time [d] E Experimental data Mathematical model Optical Density pH
Figure 12. Optical Density and pH variations, over time, and cumulative hydrogen productions,
in experiment nº2 “Run 1”, from average experimental data and from the mathematical
model by the samples: (A) B. licheniformis; (B) P. cookie; (C) Bacillus sp; (D)
Paenibacillus sp.; (E) Bacillus farraginis. The vertical bars over the experimental data
represent the standard deviations of the triplicate.
34
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
The highest hydrogen production was measured for B. farraginis (sample E), with a
total hydrogen production of 12.5 NmlH2/gVS, whereas B. licheniformis (sample A)
showed the lowest production, with a total hydrogen production of 8.3 NmlH2/gVS.
B. licheniformis and B. farraginis had a shorter lag phase than the other species, but
although this difference the yields of hydrogen were very similar to each other (see
Table 14).
An overall evaluation of all the samples, these results are much lower compared with
those obtained in experiment nº 1 – “Run 1”.
3.1.3.4 Experiment nº 2: “Run 2”
In this experiment glucose was used as substrate, with a concentration of 5 g/l, in each
sample. The results of hydrogen production potentials are reported in Table 15. Even
more, the mathematical model parameters, obtained from the average cumulative
hydrogen productions of the experimental data, are described in Table 16.
The cumulative hydrogen productions curves, from mathematical models, and the OD
and pH values obtained by the samples A, B, C, D and E are showed in Figure 13.
Table 15. Results from hydrogen production batch tests, for the experiment nº 2 – “Run 2”.
Sample
name
Hydrogen yield±SD
[Nml H2 / gVS]
Volatile Fatty Acids
Acetic acid
[mg/l]
Propionic acid
[mg/l]
Butyric acid
[mg/l]
Isovaleric acid
[mg/l]
A 76.7 ± 19.4 133.4 74.9 5.2 19.8
B 63.7 ± 16.4 144.7 5.8 4.5 14.1
C 84.5 ± 34.5 172.8 6.0 5.2 27.9
D 89.6 ± 37.8 154.3 7.2 4.4 17.5
E 95.2 ± 28.5 190.7 8.6 6.1 26.8
Table 16. Mathematical model parameters, obtained by the sampled from the experiment nº2 –
“Run 2”.
Sample
name
Exponential function parameters
P0 [Nml H2 / gVS] k [d-1
] Max rate
[(Nml H2 / gVS)*d-1
]
A 76.7 1.3 99.7
B 63.7 1.3 82.8
C 84.5 0.8 67.6
D 89.6 1.3 116.5
E 95.2 0.9 85.7
35
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2/
g V
S]
Time [d] A Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
10
20
30
40
50
60
70
80
90
0 1 2 3 4 5 6 7 8
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d] B Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6 7 8
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve h
ydro
gen
pro
du
ctio
n
[Nm
l H2
/ g
VS]
Time [d] C Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6 7 8
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d] D Experimental data Mathematical model Optical Density pH
0,000
1,000
2,000
3,000
4,000
5,000
6,000
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6 7 8
Op
tica
l D
ensi
ty /
pH
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 / g
V
S]
Time [d] E Experimental data
Mathematical model
Optical Density
pH
Figure 13. Optical Density and pH variations, over time, and cumulative hydrogen productions,
in experiment nº2 “Run 2”, from average experimental data and from the mathematical
model by the samples: (A) B. licheniformis; (B) P. cookie; (C) Bacillus sp; (D)
Paenibacillus sp.; (E) Bacillus farraginis. The vertical bars over the experimental data
represent the standard deviations of the triplicate.
36
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6 Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d]
Sample A
Sample B
Sample C
In this run the best results were obtained with the highest hydrogen productions. The
maximum value was observed in the specie B. farraginis (sample E), with a total
hydrogen production of 95.2 NmlH2/gVS. In opposite, B. licheniformis (sample A)
showed the lowest production, with a total hydrogen production of 63.7 NmlH2/gVS.
Even so, this value is higher than the values obtained in previous results.
All the samples showed similar behavior, which consists in a rapid increase in the
hydrogen production, without lag phase, until reaching a maximum value of hydrogen
production, from which the production practically ceased. At an early stage it is possible
to observe a slight increase, which subsequently decrease again. Relatively to microbial
growth, this shows a rapidly increase until achieved a maximum value.
3.2 Process performance
3.2.1 Overall evaluation of the results obtained by the pure cultures
In this section the results obtained in both experiments for the species B. licheniformis
(sample A), P. cookie (sample B) and Bacillus sp. (sample C) will be compared.
In Figure 14 it is possible to simultaneously compare the behavior, that characterizes the
hydrogen production process, obtained by the three samples in the experiment nº 1 –
“Run 1” and bellow Figure 15 shows the results obtained from all the samples analyzed
in the experiment nº 2 – “Run 1”.
Figure 14. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A, B and C in the experiment nº 1 – “Run 1”.
37
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d]
Sample A
Sample B
Sample C
Figure 15. Comparison between the cumulative hydrogen productions from the mathematical
model, obtained by the samples A, B and C in the experiment nº 2 – “Run 1”.
From both figures a similar behavior can be observed consisting into three main phases:
a lag phase, an exponential phase and a final phase.
During the first period the microorganisms were active but not yet favorable conditions
were established to have the hydrogen evolution. This means that during the lag phase,
the substrate used is mainly consumed for biomass growth. Thus, it is possible to
suppose that during the lag phase electrons mainly flow towards biosynthesis and are
not used for hydrogen evolution. The reason of this behavior remains to be explained.
The second phase is characterized by exponential gas production, during which the
medium composition changes but does not affect hydrogen production. Finally, during
the third phase, hydrogen production stops due to the low quantity of substrate that
remains in the reactor. (Ruggeri et al., 2009).
In another perspective, the hydrogen productions obtained in “Run 1”, of both
experiments, are represented in the Figure 16. In this situation, as previously mentioned,
the difference lies in the substrate used – glucose (5 g/l) was added as substrate in the
first experiment and NB (5 g/l) was added as substrate in the second experiment.
38
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
9.5 8.3
40.7
11.9 10.8 11.1
0
5
10
15
20
25
30
35
40
45
Experience nº 1 - "Run 1" Experience nº 2 - "Run 1"
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Sample A
Sample B
Sample C
Figure 16. Hydrogen yields, from “Runs 1”, obtained by the experiment nº 1 and by the
experiment nº 2.
The results demonstrate that in the experiment nº 1 – “Run 1” occurred a higher
hydrogen production by the specie P. cookie, named as sample B. This means that in
this initial phase (“Run 1”) there is a preference for the glucose, because it is an easily
biodegradable carbon source. In opposite, bacteria showed difficulty in degrade the NB,
once it is a complex substrate, for this initial state.
Despite the good result of the specie P. cookie, the other two species (B. licheniformis
and Bacillus sp.) did not show a significant hydrogen production. The main reason for
obtaining the yields lower than theoretical estimations is the utilization of the substrate
as an energy source for bacterial growth (Kapdan & Kargi, 2006).
Figure 17 displays the yields of hydrogen obtained in “Run 2” of both experiments. In
this case, the difference lies in the concentration of the substrate used. In experiment nº
1, the substrate used was glucose added with different concentrations, which were 5 g/l
and 10 g/l. On the other hand, in experiment nº 2, only 5 g/l of glucose was added.
39
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
14,8
2,7
76,7
27,9 29,4
63,7
24,0
9,9
84,5
0
10
20
30
40
50
60
70
80
90
Exp. nº 1 - "Run 2" Exp. nº 1 - "Run 2" Exp. nº 2 - "Run 2"
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Sample A
Sample B
Sample C
[C6H12O6] = 5 g/l [C6H12O6] = 10 g/l
[C6H12O6] = 5 g/l
Figure 17. Hydrogen yields, from “Runs 2”, obtained by the experiment nº 1 and by the
experiment nº 2.
The results of experiment nº1 and nº 2, with glucose concentration set to 5 g/l, show
higher hydrogen yields comparing to the experiment in which was used 10 g/l of
glucose, this means that the efficiency of the hydrogen production was decreased by
increasing glucose concentration, therefore high glucose concentration could inhibit the
fermentation process. Many studies have reported similar results regarding the
relationship of initial substrate concentration and hydrogen production (Hu et al., 2013).
It is important to consider that a proper ratio between C/N and C/P is essential for
fermentative hydrogen production. In this sense, it is possible to conclude that the
imposed substrate with the higher amount of carbon (10 g/l of glucose) supplied to the
bacteria it is not necessary for their metabolism in these proportions, because it is
inhibiting the biomass and consequently the H2 generation in the reactors and so it is
possible to minimize resources and achieve good results.
On the other hand, it was proven that the most efficient condition to obtain the higher
hydrogen production is the addition of NB as substrate, in a first run, in order to provide
the necessary nutrients for the bacteria, and subsequently add glucose as carbon source.
40
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
So, NB is providing all the nutrients that bacteria need to degrade the 5 g/l of glucose.
In other words, it represents a good balance between the nutrients available and the
substrate added. Figure 11 shows precisely these conclusions.
Therefore, can be pose pertinent questions for further researches: “How much hydrogen
could be produced if a Run 3 would be made?”; “This Run 3 will show a faster rate of
hydrogen production?”.
It seemed that the highest hydrogen yields found in this study (Experiment nº 2 – “Run
2”, see Table 15) were varied, but quite comparable to the yields of pure cultures in
other studies. For comparison, Table 17 lists hydrogen yields obtained in this work and
also from other studies that used glucose as substrate by pure cultures.
As is shown in Table 17, Clostridium and Enterobacter were most widely used as
inoculum for fermentative hydrogen production. Species of genus Clostridium are
gram-positive, rodshaped, strict anaerobes and endospore formers, whereas
Enterobacter are gram-negative, rod-shaped, and facultative anaerobes (Guo et al.,
2010).
As previously mentioned a lot of pure cultures of bacteria have been used to produce
hydrogen from various substrates. Most of the studies using pure cultures of bacteria for
fermentative hydrogen production were conducted in batch mode and used glucose as
substrate; however, it is more desirable to produce hydrogen from organic wastes using
pure cultures in continuous mode, because continuous fermentative hydrogen
production from organic wastes is more feasible for industrialization to realize the goal
of waste reduction and energy production. Thus more researches using pure cultures for
continuous fermentative hydrogen production from organic wastes are recommended
(Wang & Wan, 2009).
41
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Table 17. The pure bacterial cultures for fermentative hydrogen production. [Adapted from: Hu et
al., 2013 and Wang & Wan, 2009].
Inoculum Substrate Reactor
type
Maximum hydrogen yield
[mol H2/mol glucose] References
Bacillus sp. Glucose Batch 0.6 This study
Bacillus farraginis LF 2.7 Glucose Batch 0.7 This study
Bacillus licheniformis LF 1.33 Glucose Batch 0.6 This study
Citrobacter sp. Y 19 Glucose Batch 2.49 (Evvyernie et al., 2001)
Citrobacter amalonaticus Y19 Glucose Batch 8.7 (Oh et al., 2008)
Clostridium acetobutylicum Glucose Batch 1.8 (Lin et al., 2007)
Clostridium beijerinckii L9 Glucose Batch 2.8 (Lin et al., 2007)
Clostridium beijerinckii DSM 1820 Glucose Batch 1.5 (Masset et al., 2012)
Clostridium beijerinckii RZF – 1108 Glucose Batch 2.0 (Zhao et al., 2011)
Clostridium beijerinckii DSM 791 Glucose Batch 0.6 – 1.6 (Hu et al., 2013)
Clostridium butyricum CWBI 1009 Glucose Batch 1.7 (Masset et al., 2010)
Clostridium butyricum ATCC 19398 Glucose Batch 2.3 (Kataoka et al., 1997)
Colstridium butyricum DSM 10702 Glucose Batch 2.4 – 3.1 (Hu et al., 2013)
Clostridium paraputrificum M – 21 Glucose Batch 1.1 (Jo et al., 2008)
Clostridium pasteurianum Glucose Batch 1.5 (Ferchichi et al., 2005)
Clostridium pasteurianum DSM 525 Glucose Batch 1.8 – 3.0 (Hu et al., 2013)
Clostridium saccharoperbutylacetonicum
ATCC 27021
Glucose Batch 1.37 (Oh et al., 2003)
Enterobacter aerogenes HO – 39 Glucose Batch 1.0 (Yokoi et al., 1995)
Enterobacter aerogenes HU – 101 wt Glucose Batch 0.6 (Mahyudin et al., 1997)
Enterobacter aerogenes DSM 30053 Glucose Batch 0.1 – 0.3 (Hu et al., 2013)
Enterobacter cloacae IIT – BT 08 Glucose Batch 2.2 (Kumar & Das, 2000)
Escherichia coli MC13 – 4 Glucose Batch 1.2 (Ishikawa et al., 2006)
Escherichia coli Glucose Batch 2.0 (Bisaillon et al., 2006)
Paenibacillus sp. Glucose Batch 0.7 This study
Paenibacillus cookie LF 2.3 Glucose Batch 0.5 This study
Ruminococcus albus Glucose Batch 2.52 (Ntaikou et al., 2008)
Thermoanaerobacterium
thermosaccharolyticum KU001
Glucose Batch 2.4 (Ueno et al., 2001)
42
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
20
40
60
80
100
120
140
160
0 2 4 6 8
Cu
mu
lati
ve
hy
dro
gen
pro
du
ctio
n
[Nm
l H
2 /
g V
S]
Time [d]
Sludge 2013
Bacillus farraginis
3.2.2 Evaluation of the potential hydrogen productions from the mixed and the pure
bacteria culture
The bacteria capable of producing hydrogen widely exist in natural environments such
as soil, wastewater sludge or compost. Thus these materials can be used as inoculum for
fermentative hydrogen production. At present, the mixed cultures of bacteria from
anaerobic sludge, municipal sewage sludge, compost and soil have been widely used as
inoculum for fermentative hydrogen production (Wang & Wan, 2009).
Fermentative hydrogen production processes using mixed cultures are more practical
than those using pure cultures, because the former are simpler to operate and easier to
control, and may have a broader source of feedstock. However, in a fermentative
hydrogen production process using mixed cultures, the hydrogen produced by hydrogen
producing bacteria may be consumed by hydrogen consuming bacteria. In addition,
when mixed cultures are treated under harsh conditions, hydrogen-producing bacteria
would have a better chance than some hydrogen-consuming bacteria to survive. Thus, in
order to harness hydrogen from a fermentative hydrogen production process, the mixed
cultures can be pretreated by certain methods to suppress as much hydrogen-consuming
bacterial activity as possible while still preserving the activity of the hydrogen-
producing bacteria (Wang & Wan, 2009).
Figure 18 shows the yields of hydrogen production obtained by the samples from the
mixed and pure cultures, which achieved the best performance.
Figure 18. Cumulative hydrogen productions, from the mathematical model, obtained by the
mixed and pure bacteria cultures, which are “Sludge 2013” and Bacillus sp.,
respectively.
43
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
The highest hydrogen production was measured by the mixed bacteria culture,
represented by the sample “Sludge 2013”, with a total hydrogen production of 148.2
NmlH2/gVS. On the other hand, the pure bacteria culture, represented by Bacillus
farraginis showed a total hydrogen production of 95.2 NmlH2/gVS.
This means that the inoculum composed by the mixed bacteria culture induced to a
higher H2 production potentials. In fact, this result is expected since mixed culture is
composed by different bacterial species, with vastly different taxonomic and
physiological characteristics, which are cooperating each other allowing better results.
Nevertheless, pure culture had a good performance, since obtained a hydrogen
production only less 36% than the mixed culture. This is actually a good result, because
pure culture is composed only by one bacteria specie and so they are acting alone for
hydrogen production.
There are some factors to be taken into account to achieve good results in both
situations. The inhibition of hydrogen consumers present in the mixed cultures is
essential for hydrogen production and for further scale-up and industrial application
(Lee et al., 2011). In turn, the pure cultures can be easily contaminated by other
competitive bacteria and so the maximum sterilized conditions possible are required.
Therefore, the main purpose is to combine one pure culture with one real mixed culture
to obtain an inoculum that is a consortium of bacteria, which is characterized by the
higher potential for hydrogen production.
The vast majority of the hydrogen-producing microbial diversity however, is yet to be
discovered. This unexplored biodiversity will be tapped as more research work is
engaged in future and with setting up of mechanisms for integrated management and
utilization of these microbial resources. The potential and strategies for harnessing
microbial resources and their gene resources in dark fermentation could shed light in
further improving the yield and production rates of hydrogen fermentations (Lee et al.,
2011).
44
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
4. CONCLUSIONS
The worldwide energy need has been increasing exponentially, the reserves of fossil
fuels have been decreasing, and the combustion of fossil fuels has serious negative
effects on environment because of CO2 emission. For these reasons, many researchers
have been working on the exploration of new sustainable energy sources that could
substitute fossil fuels. In accordance with sustainable development and waste
minimization issues, bio-hydrogen gas production from renewable sources, also known
as “green technology” has received considerable attention in recent years. Therefore,
production of this clean energy source and utilization of waste materials make
biological hydrogen production a novel and promising approach to meet the increasing
energy needs as a substitute for fossil fuels (Kapdan & Kargi, 2006).
The bacteria capable of producing biological hydrogen widely exist in natural
environments and can be used as inoculum for fermentative hydrogen production. In
this study, the mixed bacteria culture used as inoculum was from an anaerobic granular
sludge. Furthermore, for the development of a specific inoculum five dark fermentation
bacteria were investigated (Bacillus licheniformis, Paenibacillus cookie, Bacillus sp.,
Paenibacillus sp. and Bacillus farraginis), in order to compared their characteristics in
hydrogen production.
The inoculum composed by the mixed bacteria culture, named as “Sludge 2013”,
induced to a higher H2 production potentials. Nevertheless, the results that Bacillus
farraginis showed were encouraging since obtained a hydrogen production only less
36% than the mixed culture.
Fermentative hydrogen production processes using mixed cultures are more practical
than those using pure cultures, because the former are simpler to operate and easier to
control, and may have a broader source of feedstock. However, in a fermentative
hydrogen production process using mixed cultures, the hydrogen produced by
hydrogen-producing bacteria may be consumed by hydrogen-consuming bacteria. The
inhibition of these last microorganisms is therefore essential for achieving good results.
Furthermore, sterilized conditions are required to obtain the best performance in the
pure cultures.
45
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Ensuring these ideal conditions, it is possible to obtain an inoculum that is a consortium
of bacteria characterized by the higher potential for hydrogen production for further
scale-up and industrial application.
In this study, the yields of hydrogen obtained by the pure bacteria, previously
mentioned, were also compared using two types of substrates (glucose and NB) and
changing the initial concentration in glucose.
The results showed that the efficiency of the hydrogen production was decreased by
increasing glucose concentration, therefore high glucose concentration could inhibit the
fermentation process. Moreover, it was proven that the most efficient condition to
obtain the higher hydrogen production is the addition of NB as substrate, in a first run,
in order to provide the necessary nutrients for the bacteria, and subsequently add
glucose as carbon source.
The objectives initially proposed for this study were achieved. Thus, further research is
necessary to better understand the impact of the composition of the substrate on
biological hydrogen performances.
Therefore, future investigations may be interesting, taking into account the following
pertinent questions: I) “How much hydrogen could be produced using pure cultures if a
further run would be made adding new substrate?”, II) “Will this further run show a
faster rate of hydrogen production compared to previous?” and III) “What is the best
combination of pure culture species to achieve the highest hydrogen yields?”.
46
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
REFERENCES
Alibardi L., Favaro L., Lavagnolo M. C., Basaglia M., Casella S. & Cossu R. (2009).
Microbiological analyses in batch test for hydrogen production. In: Sardinia 2009
Twelfth International Waste Management and Landfill Symposium. CISA, Italy.
Alibardi L., Favaro L., Lavagnolo M. C., Basaglia M. & Casella S. (2012). Effects of
heat treatment on microbial communities of granular sludge for biological
hydrogen production. Water Science & Technology 66: 1483-1490.
APHA, AWWA, WPCF (1999) Standard Methods for the Examination of Water and
Wastewater. 20th edition, American Public Health Association, American Water
Works Association, Water Environment Federation, Washington, DC.
Bisaillon A., Turcot J. & Hallenbeck P. C. (2006). The effect of nutrient limitation on
hydrogen production by batch cultures of Escherichia coli. International Journal
of Hydrogen Energy 31: 1504 – 1508.
Debabrata D. & Nejat V. T. (2001). Hydrogen production by biological processes: a
survey of literature. International Journal of Hydrogen Energy 26: 13 – 28.
Evvyernie D., Morimoto K., Karita S., Kimura T., Sakka K. & Ohmiya K. (2001).
Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum
M-21. Journal of Bioscience and Bioengineering 91: 339 – 343.
Fan Y.T., Li C.L., Lay J.J., Hou H.W. & Zhang G.S. (2004). Optimization of initial
substrate and pH levels for germination of sporing hydrogen-producing anaerobes
in cow dung compost. Bioresource Technololy 91: 189–193.
Fang H. H. P. & Liu H. (2002). Effect of pH on hydrogen production from glucose by a
mixed culture. Bioresource Technololy 82: 87-93.
Favaro L., Alibardi L., Lavagnolo M. C., Basaglia M., Cossu R. & Casella S. (2011).
Looking for robust and eficiente H2-production microbes. In: Sardinia 2011
Thirteenth International Waste Management and Landfill Symposium. CISA,
Italy.
Favaro L., Alibardi L., Lavagnolo M. C., Casella S. & Basaglia M. (2013). Effects of
inoculum and indigenous microflora on hydrogen production from the organic
fraction of municipal solid waste. International Journal of Hydrogen Energy. In
press.
Ferchichi M., Crabbe E., Hintz W., Gil G. H. & Almadidy A. (2005). Influence of
culture parameters on biological hydrogen production by Clostridium
saccharoperbutylacetonicum ATCC 27021. World Journal of Microbiology &
Biotechnology 21: 855 – 862.
47
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Guo X. M., Trably E., Latrille E., Carrère H. & Steyer J. P. (2010). Hydrogen
production from agricultural waste by dark fermentation: a review. International
Journal of Hydrogen Energy 35: 10660 – 10673.
Hallenbeck P. C. & Benemann J. R. (2002). Biological hydrogen production:
fundamentals and limiting processes. International Journal of Hydrogen Energy
27: 1185 – 1193.
Hawkes F. R., Dinsdale R., Hawkes D. L., Hussy I. (2002). Sustainable fermentative
hydrogen production: challenges for process optimization. International Journal
of Hydrogen Energy 27: 1339 – 1347.
Hu C. C., Giannis A., Chen C. L., Qi W. & Wang J. Y. (2013). Comparative study of
biohydrogen production by four dark fermentative bacteria. International Journal
of Hydrogen Energy: 1 – 7. In press.
Ishikawa M., Yamamura S., Takamura Y., Sode K., Tamiya E. & Tomiyama M. (2006).
Development of a compact high-density microbial hydrogen reactor for portable
bio-fuel cell system. International Journal of Hydrogen Energy 31: 1484 – 1489.
Jo J. H., Lee D. S., Park D., Choe W. S. & Park J. M. (2008). Optimization of key
process variables for enhanced hydrogen production by Enterobacter aerogenes
using statistical methods. Bioresource Technology 99: 2061 – 2066.
Kalia V. C. & Purohit H. J. (2008). Microbial diversity and genomics in aid of
bioenergy. Journal of industrial microbiology & biotechnology 35: 403 – 419.
Kapdan I. K. & Kargi F. (2006). Bio-hydrogen production from waste material. Enzyme
and Microbial Technology 38: 569 – 582.
Kataoka N., Miya K. & Kiriyama K. (1997). Studies on hydrogen production by
continuous culture system of hydrogen-production anaerobic bacteria. Water
Science & Technology 36: 41-47.
Kumar N. & Das D. (2000). Enhancement of hydrogen production by Enterobacter
cloacae IIT – BT 08. Process Biochemistry 35: 589 – 593.
Lay J. J., Li Y. Y. & Noike T. (1997). The influences of pH and moisture content on the
methane production in high-solids sludge digestion. Water Research 31: 1518 –
24.
Lee D. J., Show K. Y. & Su A. (2011). Dark fermentation on biohydrogen production:
pure culture. Bioresource Technology 102: 8393 – 8402.
Lin P. Y., Whang L. M., Wu Y. R., Ren W. J. & Hsiao C. J. et al. (2007). Biological
hydrogen production of the genus Clostridium: metabolic study and mathematical
model simulation. International Journal of Hydrogen Energy 32: 1728 – 1735.
48
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Mahyudin A. R., Furutani Y., Nakashimada Y., Kakizono T. & Nishio N. (1997).
Enhanced hydrogen production in altered mixed acid fermentation of glucose by
Enterobacter aerogenes. Journal of Fermentation & Bioengineering 83: 358 –
363.
Maintinguer S. I., Fernandes B. S., Duarte I. C. S., Saavedra N. K., Adorno M. A. &
Varesche M. B. (2008). Fermentative hydrogen production by microbial
consortium. International Journal of Hydrogen Energy 33: 4309 – 4317.
Masset J., Hiligsmann S., Hamilton C., Beckers L., Franck F. & Thonart P. (2010).
Effect of pH on glucose and starch fermentation in batch and sequenced batch
mode with a recently isolated strain of hydrogen-producing Clostridium
butyricum CWBI1009. International Journal of Hydrogen Energy 35: 3371 –
3378.
Masset J., Calusinska M., Hamilton C., Hiligsmann S., Joris B., Wilmotte A., et al.
(2012). Fermentative hydrogen production from glucose and starch using strains
and artificial co-cultures of Clostridium spp. Biotechnology Biofuels 5: 35.
McCarty P. L. (1964). Anaerobic waste treatment fundamentals. Stanford University.
Miller G. L. (1959). Use of dinitrosalicyclic acid reagent for determination of reducing
sugars. Analytical Chemistry 31: 426 – 428.
Ni M., Leung D. Y. C., Leung M. K. H. & Sumathy K. (2006). An overview of
hydrogen production from biomass. Fuel Processing Technology 87: 461 – 472.
Ntaikou I., Gavala H. N., Kornaros M. & Lyberatos G. (2008). Hydrogen production
from sugars and sweet sorghum biomass using Ruminococcus albus. International
Journal of Hydrogen Energy 33: 1153 – 1163.
Oh Y. K., Seol E. H., Kim J. R. & Park S. (2003). Fermentative biohydrogen production
by a new chemoheterotrophic bacterium Citrobacter sp. Y19. International
Journal of Hydrogen Energy 28: 1353 – 1359.
Oh Y. K., Kim H. J., Park S., Kim M. S. & Ryu D. D. Y. (2008). Metabolic-flux
analysis of hydrogen production pathway in Citrobacter amalonaticus Y19.
International Journal of Hydrogen Energy 33: 1471 – 1482.
Patel S. K. S., Kumar P. & Kalia V. C. (2012). Enhancing biological hydrogen
production through complementary microbial metabolisms. International Journal
of Hydrogen Energy 37: 10590 – 10603.
Ruggeri B., Tommasi T. & Sassi G. (2009). Experimental kinetics and dynamics of
hydrogen production on glucose by hydrogen forming bacteria (HFB) culture.
International Journal of Hydrogen Energy 34: 753 – 763.
49
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Ting C. H., Lin K. R., Lee D. J. & Tay J. H. (2004). Production of hydrogen and
methane from wastewater sludge using anaerobic fermentation. Water Science &
Technology 50 (9): 223-228.
Tommasi T., Sassi G. & Ruggeri B. (2008). Acid pre-treatment of sewage anaerobic
sludge to increase hydrogen producing bacteria HPB: effectiveness and
reproducibility. Water Science & Technology 58 (8): 1623-1628.
Trzcinski A. P. & Stuckey D. C. (2012). Determination of the hydrolysis constant in the
biochemical methane potential test of municipal solid waste. Environmental
Engineering Science 29(9): 848 – 854.
Ueno Y., Haruta S., Ishii M. & Igarashi Y. (2001). Characterization of a microorganism
isolated from the effluent of hydrogen fermentation by microflora. Journal of
Bioscience & Bioengineering 92: 397 – 400.
Van Ginkel S. & Sung S. (2001). Biohydrogen production as a function of pH and
substrate concentration. Environmental Science Technology 35: 4726-4730.
Van Ginkel S. W., Oh S. E. & Logan B. E. (2005). Biohydrogen gas production from
food processing and domestic wastewater. International Journal of Hydrogen
Energy 30: 1535 – 1542.
Vavilin V. A., Rytow S. V. & Lokshina L. Y. (1995). Modelling hydrogen partial
pressure change as a result of competition between the butyric and propionic
groups of acidogenic bacteria. Bioresource Technology 54: 171 – 177.
Vijayaraghavan K. & Soom M. A. M. (2004). Trends in biological hydrogen
production: a review. International Association of Hydrogen Energy.
Wang J. & Wan W. (2009). Factors influencing fermentative hydrogen production: A
review. International Journal of Hydrogen Energy 34: 799 – 811.
Yokoi H., Ohkawara T., Hirose J., Hayashi S. & Takasaki Y. (1995). Characteristics of
hydrogen production by aciduric Enterobacter aerogenes strain HO – 39. Journal
of Fermentation & Bioengineering 80: 571 – 574.
Zhao X., Xing D., Fu N., Liu B., Ren N. & Zhao X. (2011). Hydrogen production by
the newly isolated Clostridium beijerinckii RZF – 1108. Bioresource Technology
102: 8432 – 8436.
50
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
ANNEX 1
Results of the BMP tests
Initially, batch tests were conducted for the evaluation of the sequential production of
hydrogen and methane from the selected three specific types of granular sludge. The
second phase of methane production was performed as soon as hydrogen production
lasted.
For the BMP tests preparation, 50 g of granular sludge from 2013 were added in all the
batch reactors available. Even so, the samples remained with the same identification:
“Sludge 2011”, “Sludge 2012” and “Sludge 2013”.
The sludge from 2013 was selected because it showed, in the first part of this work, the
best performance in terms of hydrogen production rate. It is important to note that the
granular sludge used was not pre-treated prior to starting the experimental tests as
inoculum of batch test for CH4 production.
Moreover, to provide optimal conditions for methanogenic bacteria, the pH of the
digestion liquid was raised from 5.5 to 7.5 by adding Na2CO3.
Anaerobic conditions were obtained by making nitrogen flow trough the head space of
the vessel for 3 minutes. After this operation the excess pressure was removed in order
to re-establish the atmospheric pressure. The mesophilic conditions were guaranteed by
keeping the reactors in a water bath at a steady temperature of 35° C (± 1° C).
The amount of biogas produced was recorded daily, using the water displacement
method and biogas composition in terms of hydrogen, carbon dioxide and methane was
measured by a gas chromatograph, in this order quantity and the quality of the biogas
were measured. The BMP tests took place over 45 days, after which no longer
significant production of methane was noted.
51
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
Since an opportunity to establish an internal co-operation with colleagues from the
Department of Agronomy Food Natural Resources Animals and Environment of the
University of Padova came up, the hydrogen production became the main theme and so
at the end of the BMP tests the experimental data were treated, in order to understand
the behavior that each sample had at the second stage of the anaerobic digestion
process. These results were used for comparison with other BMP tests results, obtained
by a student who was doing her master’s thesis in the laboratory. Furthermore these
results can also be useful for future investigations in this field.
The results from methane production batch tests obtained by each sample and the
mathematical model parameters, obtained from the average cumulative methane
productions of the experimental data, are described in the following table.
Sample name
Methane
yield±SD [Nml
CH4 / gVS]
Exponential function parameters
P0
[Nml CH2 / gVS]
k
[d-1
]
Max rate
[(Nml H2 / gVS)*d-1
]
Sludge 2011 384.1 ± 152.2 384.1 0.08 30.7
Sludge 2012 325.6 ± 31.3 325.6 0.10 32.6
Sludge 2013 407.8 ± 41.8 407.8 0.04 16.3
In the graphs above are represented the cumulative methane productions curves from
experimental data average and from the mathematical model obtained by the three
samples: (I) “Sludge 2011”; (II) “Sludge 2012”; (III) “Sludge 2013”. The vertical bars
over the experimental data represent the standard deviations of the triplicate.
52
Biological Hydrogen Production using Organic Waste and Specific Bacterial Species
0
100
200
300
400
500
600
0 10 20 30 40 50
Cu
mu
lati
ve
met
ha
ne
pro
du
ctio
n
[Nm
l C
H4 / g
V
S]
Time [d]
Experimental data
Mathematical model
0
50
100
150
200
250
300
350
400
0 10 20 30 40 50
Cu
mu
lati
ve
met
ha
ne
pro
du
ctio
n
[Nm
l C
H4 /
g V
S]
Time [d]
Experimental data
Mathematical model
II
0
50
100
150
200
250
300
350
400
450
500
0 10 20 30 40 50
Cu
mu
lati
ve m
eth
an
e p
ro
du
cti
on
[Nm
l C
H4 /
g V
S]
Time [d]
Experimental data
Mathematical model
III
I
53